- Volume 82, Issue 6, 2001
Volume 82, Issue 6, 2001
- Animal: RNA Viruses
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Increased proteolytic activity and matrix metalloprotease expression in lungs during infection by porcine reproductive and respiratory syndrome virus
More LessThe local increase in the secretion of extracellular proteases, allowing cleavage of the extracellular matrix and thereby facilitating the infiltration of T cells, monocytes and neutrophils, is a hallmark of chronic inflammation and autoimmunity. In pulmonary genetic diseases, such as emphysema and cystic fibrosis, proteases can also favour the development of local immunodeficiency by degrading key regulators of the immune response, such as CD4, CD8, IgG, ICAM-1 and C3b receptors. Since several infectious agents can give rise to severe pulmonary disorders associated with opportunistic infections, we sought to determine whether an increase in proteolytic activity occurred during infection with porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of a new disease in swine characterized by severe respiratory problems in young pigs. Piglets were infected with the virus and bronchoalveolar lavages were collected at various times post-infection to measure the net proteolytic activity. It was shown that PRRSV infection leads to a significant increase in proteolytic activity in pulmonary fluids. Maximal activity was found at 7 and 14 days post-infection, with a return towards normal levels at day 42. Zymographic analyses showed a significant increase in the secretion of matrix metalloproteases (MMPs) 2 and 9, two enzymes involved in tissue remodelling. Histological analyses showed a correlation between the increase in proteolytic activity and the appearance of lesions that were characterized by massive lymphomononuclear cell infiltration. These results suggest that virus infection of the lungs can lead to a transient increase in proteolytic activity that could favour opportunistic infection.
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Reversion of a live porcine reproductive and respiratory syndrome virus vaccine investigated by parallel mutations
A live attenuated porcine reproductive and respiratory syndrome (PRRS) vaccine virus has been shown to revert to virulence under field conditions. In order to identify genetic virulence determinants, ORF1 from the attenuated vaccine virus and three Danish vaccine-derived field isolates was sequenced and compared with the parental strain of the vaccine virus (VR2332). This revealed five mutations that had occurred independently in all three vaccine-derived field isolates, indicating strong parallel selective pressure on these positions in the vaccine virus when used in swine herds. Two of these parallel mutations were direct reversions to the parental VR2332 sequence and were situated in a papain-like cysteine protease domain and in the helicase domain. The remaining parallel mutations might be seen as second-site compensatory mutations for one or more of the mutations that accumulated in the vaccine virus sequence during cell-culture adaptation. Evaluation of the remaining mutations in the ORF1 sequence revealed stronger selective pressure for amino acid conservation during spread in pigs than during vaccine production. Furthermore, it was found that the selective pressure did not change during the time period studied. The implications of these findings for PRRS vaccine attenuation and reversion are discussed.
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Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus
More LessThe coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reverse-genetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.
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Evidence for recombination in natural populations of dengue virus type 1 based on the analysis of complete genome sequences
Recombination events are known to occur in non-segmented RNA viruses like polioviruses or alphaviruses. Analysis of the subgenomic sequences of dengue virus type 1 (DENV-1) structural genes has recently allowed the identification of possible recombination breakpoints. Because DENV is a major human pathogen, this discovery might have important implications for virus pathogenicity, vaccine safety and efficiency, or diagnosis and, therefore, requires clear confirmation. We report the complete sequence determination of one Asian and two African strains of DENV-1 isolated from human patients. Rigorous sequence analysis provided strong evidence for the occurrence of intragenomic recombination events between DENV-1 strains belonging to different lineages. Singapore S275/90 strain appears to be the evolutionary product of a recombination event between viruses belonging to two distinct lineages: one lineage includes an African strain isolated in Abidjan (Ivory Coast) and the other includes isolates from Djibouti and Cambodia. The ‘Recombination Detection Program’, bootscanning and analysis of diversity plots provided congruent results concerning the existence of a two-switch recombination event and the localization of recombination breakpoints. Thus, the 5′ and 3′ genomic ends of the Singapore S275/90 strain were inherited from a Djibouti/Cambodia lineage ancestor and an internal fragment located in the envelope/NS1 region originated from an Abidjan lineage ancestor.
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Infectious cDNA clone of the hepatitis C virus genotype 1 prototype sequence
More LessA full-length cDNA clone of the hepatitis C virus (HCV) genotype 1 prototype (subtype 1a) sequence was constructed. Synthetic RNA produced from the initial cDNA clone was not infectious following intrahepatic inoculation of a chimpanzee. A consensus clone was prepared by comparison with multiple full-length HCV sequences of genotypes 1, 2 and 3. A total of 11 non-consensus amino acid residues were altered by mutagenesis. Synthetic RNA from the repaired clone initiated a typical, acute-resolving HCV infection following intrahepatic inoculation of a chimpanzee. In addition, at least one of three chimeric cDNA clones constructed between the HCV-1 and H77 genotype 1a strains of HCV was infectious in a chimpanzee. This is the first example of an infectious chimeric HCV clone. An infectious cDNA clone of HCV-1 will be of particular value, since it is the prototype HCV sequence and many commonly used reagents are based on this sequence.
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Humoral and CD4+ T helper (Th) cell responses to the hepatitis C virus non-structural 3 (NS3) protein: NS3 primes Th1-like responses more effectively as a DNA-based immunogen than as a recombinant protein
The non-structural 3 (NS3) protein is one of the most conserved proteins of hepatitis C virus, and T helper 1 (Th1)-like responses to NS3 in humans correlate with clearance of infection. Several studies have proposed that DNA-based immunizations are highly immunogenic and prime Th1-like responses, although few head-to-head comparisons with exogenous protein immunizations have been described. A full-length NS3/NS4A gene was cloned in eukaryotic vectors with expression directed to different subcellular compartments. Inbred mice were immunized twice in regenerating tibialis anterior (TA) muscles with either plasmid DNA or recombinant NS3 (rNS3). After two 100 μg DNA immunizations, specific antibody titres of up to 12960 were detected at week 5, dominated by IgG2a and IgG2b. NS3-specific CD4+ T cell responses in DNA-immunized mice peaked at day 13, as measured by proliferation and IL-2 and IFN-γ production. Mice immunized with 1–10 μg rNS3 without adjuvant developed antibody titres comparable to those of the DNA-immunized mice, but dominated instead by IgG1. CD4+ T cell responses in these mice showed peaks of IL-2 response at day 3 and IL-6 and IFN-γ responses at day 6. With adjuvant, rNS3 was around 10-fold more immunogenic with respect to speed and magnitude of the immune responses. Thus, immunization with rNS3 in adjuvant is superior to DNA immunization with respect to kinetics and quantity in priming specific antibodies and CD4+ T cells. However, as a DNA immunogen, NS3 elicits stronger Th1-like immune responses, whereas rNS3 primes a mixed Th1/Th2-like response regardless of the route, dose or adjuvant.
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Induction of apoptosis in bone marrow neutrophil-lineage cells by classical swine fever virus
More LessThe pathogenesis of bone marrow atrophy during classical swine fever (CSF) was investigated in vitro by using CSF virus (CSFV) infection of bone marrow haematopoietic cells (BMHC). The monocytic lineage had the highest susceptibility to CSFV infection, whereas the more mature SWC8+ granulocytic cells were not directly susceptible to infection. However, myelomonocytic precursors were targets for CSFV infection and continued to differentiate into SWC8+ granulocytic cells, which remained infected. This explains the occurrence of infected peripheral blood granulocytes during CSF. The infection of BMHC resulted in increased apoptosis and necrosis, mainly within the granulocytic lineage. Caspases 3 and 9 were particularly activated, relating to the mitochondrial pathway of apoptosis. Interestingly, the majority of infected cells were non-apoptotic, the apoptotic cells being primarily non-infected. This indicated an indirect mechanism for induction of apoptosis, but no role could be identified for bone marrow stroma cells such as macrophages or fibroblastoid cells. Furthermore, soluble factors including cytokines and reactive oxygen species were not primarily responsible. In contrast, contact between infected and non-infected BMHC was critical for increasing apoptosis in the latter. Taken together, these results in vitro relate to and help to explain further the apoptosis of BMHC that occurs in vivo during CSF. This experimental system will also be particularly useful for the study of CSFV gene products involved in leukocyte apoptosis.
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Distribution and characterization of tick-borne encephalitis viruses from Siberia and far-eastern Asia
In this study, tick-borne encephalitis (TBE) viruses from Siberia and far-eastern Asia were characterized in order to determine virus subtype distribution. TBE viruses were isolated from ticks (Ixodes persulcatus) collected in the far-eastern (Khabarovsk and Vladivostok) and Siberian (Irkutsk) regions of Russia in 1999. Phylogenetic analysis showed that isolates formed distinct clusters of far-eastern and Siberian subtypes. There was also a minor difference in antigenicity between the Irkutsk isolates and other TBE virus strains, as demonstrated by the reactivity of monoclonal antibodies. Amino acid alignments of the E gene showed that the Irkutsk isolates had a single amino acid change at position 234 (Q or H); this amino acid position is considered to be a ‘signature’ of Siberian subtype TBE viruses. Strains isolated in Irkutsk also exhibited equivalent or somewhat higher virulence in mice compared with far-eastern TBE virus isolates. All viruses isolated in this study (i.e. far-east Asian and Siberian isolates) have 3′ non-coding regions (NCRs) of almost the same length, which contrasts with the various sizes of 3′NCRs of other TBE viruses strains reported previously. The data presented in this study show that the 3′NCR is uniform among TBE viruses isolated from Siberia and far-eastern Asia and that the 3′NCR is essential for TBE virus growth in tick and/or rodent host cells.
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Sabin attenuated LSc/2ab strain of poliovirus spreads to the spinal cord from a peripheral nerve in bonnet monkeys (Macaca radiata)
More LessVaccine-associated paralytic poliomyelitis is a serious concern while using the live attenuated oral polio vaccine for the eradication of poliomyelitis. The bonnet monkey model of poliovirus central nervous system (CNS) infection following experimental inoculation into the ulnar nerve allows the comparative study of wild-type and attenuated poliovirus invasiveness. Dosages ⩾104 TCID50 of Mahoney strain of poliovirus type 1 [PV1(M)] result in paralysis. In contrast, even with 107 TCID50 of Sabin attenuated strain of poliovirus type 1 (LSc/2ab), no paralysis occurs, but virus spreads into the CNS where viral RNA is found in spinal cord neurons. While wild-type PV1(M) viral RNA replicates in neurons (and possibly in glial cells) and in cells around vessel walls, which may be mononuclear or endothelial cells, attenuated viral RNA is detected only in neurons. Systemic viraemia and gastrointestinal virus shedding occurs only in PV1(M)-infected animals. While a systemic serologic response is detected in both groups of animals, cerebrospinal fluid antibodies are detected only in animals infected with PV1(M). Both the PV1(M) and LSc/2ab strains spread to the cervical spinal cord and then to the lumbar spinal cord following ulnar nerve inoculation. Neuronophagia and neuronal loss are only seen in PV1(M)-infected monkeys in whom clinical paralysis is observed. Infection with LSc/2ab does not result in neuronophagia, neuronal loss or clinical paralysis. Spread of attenuated poliovirus in spinal cord neurons without causing paralysis following inoculation into the ulnar nerve is an important finding.
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Genetic characterization of the coxsackievirus B2 3′ untranslated region
More LessThe secondary structure of the 3′ untranslated region (3′UTR) of picornaviruses is thought to be important for the initiation of negative-strand RNA synthesis. In this study, genetic and biological analyses of the 3′ terminus of coxsackievirus B2 (CVB2), which differs from other enteroviruses due to the presence of five additional nucleotides prior to the poly(A) tail, is reported. The importance of this extension was investigated using a 3′UTR mutant lacking the five nucleotides prior to the poly(A) tail and containing two point mutations. The predicted secondary structure within the 3′UTR of this mutant was less energetically favourable compared with that of the wild-type (wt) genotype. This mutant clone was transfected into green monkey kidney cells in four parallel experiments and propagated for multiple passages, enabling the virus to establish a stable revertant genotype. Genetic analysis of the virus progeny from these different passages revealed two major types of revertant. Both types showed wt-like growth properties and more stable and wt-like predicted secondary structures than the parent mutant clone. The first type of revertant neutralized the introduced point mutation with a compensatory second-site mutation, whereas the second type of revertant partly compensated for the deletion of the five proximal nucleotides by the insertion of nucleotides that matched the wt sequence. Therefore, the extended 3′ end of CVB2 may be considered to be a stabilizing sequence for RNA secondary structure and an important feature for the virus.
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The role of IL-5, IL-6 and IL-10 in primary and vaccine-primed immune responses to infection with Friend retrovirus (Murine leukaemia virus)
More LessThe defence of a host against viral infections is strongly influenced by cytokines. We investigated the role of the B-cell stimulating cytokines IL-5 and IL-6, and the immuno-suppressive cytokine IL-10, during primary and secondary immune responses in mice against infection with Friend retrovirus (FV) (Murine leukaemia virus). IL-5−/− mice were comparable to C57BL/6 wild-type mice in their ability to control acute FV infection. In contrast, IL-6−/− and IL-10−/− mice showed significantly enhanced virus loads in spleen cells. However, this impaired control of acute FV replication did not alter the long-term control over persistent FV in IL-6−/− and IL-10−/− mice. Immunization with a live attenuated vaccine virus prior to challenge protected all three types of cytokine-deficient mice from high levels of spleen virus, despite the finding that the vaccinated IL-5- and IL-6-deficient mice had significantly reduced titres of virus-neutralizing IgG class antibodies. The results indicate that IL-6 and IL-10 contribute to primary immune responses against FV, but are dispensable during persistent infection and vaccine-primed secondary responses.
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Jaagsiekte sheep retrovirus can be detected in the peripheral blood during the pre-clinical period of sheep pulmonary adenomatosis
Peripheral blood leukocytes (PBLs) and tissue samples from 36 sheep were examined for jaagsiekte sheep retrovirus (JSRV) by hemi-nested PCR. Animals were classified according to the status of sheep pulmonary adenomatosis (SPA), which was confirmed by pathological examination, as follows: (i) sheep with classical SPA (cSPA, n=10), (ii) sheep with atypical SPA (aSPA, n=6), (iii) non-affected sheep from SPA-affected flocks (in-contact, n=10) and (iv) non-affected sheep from SPA-free flocks (control, n=10). JSRV proviral DNA was detected in the PBLs of 10/10 cSPA, 5/6 aSPA, 4/10 in-contact and 0/10 control sheep. Lung tumours and lymphoid organs were also found to be JSRV-positive. The number of positive PCR results was greater for sheep in the cSPA group than for those in the aSPA and in-contact groups. For the first time, it is concluded that JSRV can be detected in naturally infected sheep before the onset of clinical disease and even before the development of discernible tumours.
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Retroviral recombination is temperature dependent
Ting Li and Jiayou ZhangTwo conflicting in vitro observations suggest that retroviral recombinations are temperature dependent. Ouhammouch & Brody (Nucleic Acids Research 20, 5443–5450, 1992) suggested that retroviral recombination rates should increase as temperature increases. However, Shimomaye & Salvato (Gene Analysis Techniques 6, 25–28, 1989) and Brooks et al. (Biotechniques 19, 806–812, 814–815, 1985) found that at low temperature the tightly folded structure of RNAs may hinder reverse transcription proceeding along the RNA template, which increases its chance of dissociating from the template; therefore, raising the reaction temperature was the simplest way to overcome template secondary structure and prevent premature termination of cDNA synthesis. In this report, two vectors based on murine leukaemia virus (MLV) were constructed. The first contained two mutated gfp genes in tandem positions. The upstream gfp gene encoded a mutation at its 3′ end, while the downstream gfp gene encoded a mutation at its 5′ end. The recombination that occurred between the two mutated gfp genes restored a functional gfp gene. The cells that contained the functional gfp gene were green when observed under a fluorescence microscope. The second MLV vector contained a functional gfp gene with two identical sequences flanking either end. A recombination that occurred between the two identical sequences resulted in deletion of the gfp gene. Cells containing the vector with the gfp deletion were colourless or clear when observed under the microscope. Using these two vectors, we have demonstrated that retroviral recombination is temperature dependent and the rate of recombination decreases as temperature is raised from 31 to 43 °C.
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The role of the Type I interferon response in the resistance of mice to filovirus infection
More LessAdult immunocompetent mice inoculated with Ebola (EBO) or Marburg (MBG) virus do not become ill. A suckling-mouse-passaged variant of EBO Zaire ’76 (‘mouse-adapted EBO-Z’) causes rapidly lethal infection in adult mice after intraperitoneal (i.p.) inoculation, but does not cause apparent disease when inoculated subcutaneously (s.c.). A series of experiments showed that both forms of resistance to infection are mediated by the Type I interferon response. Mice lacking the cell-surface IFN-α/β receptor died within a week after inoculation of EBO-Z ’76, EBO Sudan, MBG Musoke or MBG Ravn, or after s.c. challenge with mouse-adapted EBO-Z. EBO Reston and EBO Ivory Coast did not cause illness, but immunized the mice against subsequent challenge with mouse-adapted EBO-Z. Normal adult mice treated with antibodies against murine IFN-α/β could also be lethally infected with i.p.-inoculated EBO-Z ’76 or EBO Sudan and with s.c.-inoculated mouse-adapted EBO-Z. Severe combined immunodeficient (SCID) mice became ill 3–4 weeks after inoculation with EBO-Z ’76, EBO Sudan or MBG Ravn, but not the other viruses. Treatment with anti-IFN-α/β antibodies markedly accelerated the course of EBO-Z ’76 infection. Antibody treatment blocked the effect of a potent antiviral drug, 3-deazaneplanocin A, indicating that successful filovirus therapy may require the active participation of the Type I IFN response. Mice lacking an IFN-α/β response resemble primates in their susceptibility to rapidly progressive, overwhelming filovirus infection. The outcome of filovirus transfer between animal species appears to be determined by interactions between the virus and the innate immune response.
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Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells
More LessThe intracellular cleavage of respiratory syncytial virus (RSV) fusion (F) protein by furin was examined. In RSV-infected LoVo cells, which express an inactive form of furin, and in RSV-infected Vero cells treated with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk), the F protein was expressed as a non-cleaved 73 kDa species. In both cases the F protein was initially expressed as an endoglycosidase H (Endo H)-sensitive precursor (F0EHs) which was modified approximately 40 min post-synthesis by the addition of complex carbohydrates to produce the Endo H-resistant form (F0EHr). The size and glycosylation state of F0EHr were identical to a transient intermediate form of non-cleaved F protein which was detected in RSV-infected Vero cells in the absence of inhibitor. Cell surface biotinylation and surface immunofluorescence staining showed that F0EHr was present on the surface of RSV-infected cells. RSV filaments have been shown to be the predominant form of the budding virus that is detected during virus replication. Analysis of the RSV-infected cells using scanning electron microscopy (SEM) showed that, in the presence of dec-RVKR-cmk, virus budding was impaired, producing fewer and much smaller viral filaments than in untreated cells. A comparison of immunofluorescence and SEM data showed that F0EHr was routed to the surface of virus-infected cells but not located in these smaller structures. Our findings suggest that activation of the F protein is required for the efficient formation of RSV filaments.
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A haemagglutinin (HA1)-specific FAb neutralizes influenza A virus by inhibiting fusion activity
More LessH9-D3-4R2 (referred to as H9), a murine monoclonal HA1-specific IgG3, recognizes an epitope within antigenic site Cb of influenza virus A/PR/8/34 (H1N1). At 50% neutralization, inhibition of virus-mediated fusion was responsible for the majority of neutralization but, at higher antibody concentrations, the attachment of virus to target cells was also inhibited and may have contributed to neutralization. H9 FAb was also neutralizing, although the concentration needed was two orders of magnitude greater than for the IgG. Functional affinity of the IgG and affinity of the FAb were almost identical, and it is not clear why the neutralization efficiency of the FAb was so low. Unlike its IgG, H9 FAb had no detectable effect on virus attachment but inhibited virus fusion activity. It thus appears that monovalent binding by this antibody is sufficient to inhibit fusion activity and that this was directly responsible for neutralization of infectivity.
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Infection of a child in Hong Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs
V. Gregory, W. Lim, K. Cameron, M. Bennett, S. Marozin, A. Klimov, H. Hall, N. Cox, A. Hay and Y. P. LinInfluenza virus A/Hong Kong/1774/99, isolated from a young child with mild influenza, was shown to be similar in its antigenic and genetic characteristics to H3N2 viruses circulating in pigs in Europe during the 1990s and in particular to be closely related to viruses isolated from two children in the Netherlands in 1993. Similar viruses had previously not been identified outside Europe. Although there is little evidence as to how the child contracted the infection, it appears likely that pigs in southern China were the source of infection. Characteristics shared with the European swine viruses include resistance to the anti-influenza drugs amantadine and rimantadine. Thus not only does this incident once again highlight the potential of pigs as a source of novel human influenza viruses, but also indicates the potential for emergence of amantadine-resistant human viruses.
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- Animal: DNA Viruses
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Characterization and functional analysis of Serp3: a novel myxoma virus-encoded serpin involved in virulence
Myxoma virus (MV), a member of the family Poxviridae, is the causative agent of myxomatosis, a fatal disease of the European rabbit. The MV genome is a linear, double-stranded DNA molecule that encodes several factors important for evasion of the host immune system. Sequencing the right-end region of the MV genome identified an 801 bp open reading frame (ORF) encoding a polypeptide that belongs to the serpin superfamily. To date, two MV-encoded serpins have been characterized: SERP-1 binds to several targets and is an anti-inflammatory molecule, whereas Serp2 is essential for virus virulence and has both anti-inflammatory and anti-apoptotic effects. Thus, Serp3 is the third MV-encoded serpin. DNA sequence analysis of Serp3 indicated a similarity to poxvirus late promoters, which was confirmed by mRNA expression analysis. Serp3 has an atypical serpin motif and has significant sequence deletions as compared to most cellular and viral serpins. However, molecular modelling studies suggested that Serp3 can retain the overall serpin fold. Insertional inactivation of the serp3 ORF led to a significant attenuation of virulence in vivo (as measured by the increase in survival of infected rabbits) and limited dissemination of the virus to secondary sites of infection. In rabbits infected with a Serp3 deletion mutant (MV-Serp3−), the main histopathological feature is the absence of secondary myxomas. Both wild-type MV and MV-Serp3− replicate at comparable levels in vivo. Serp3 may represent a significant virulence factor of MV and probably acts in synergy with other viral proteins.
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Plasma membrane requirements for cell fusion induced by herpes simplex virus type 1 glycoproteins gB, gD, gH and gL
More LessHerpes simplex virus type 1 (HSV-1) glycoproteins gB, gD and gHL are capable of inducing cell fusion when expressed from plasmid vectors in the absence of any other virus components. Fusion requires the expression of all four glycoproteins on the same membrane, since they are unable to cooperate in trans to induce syncytium formation. In addition, the fusion event is dependent on the expression of a gD receptor on target cell membranes and does not require the presence of cell-surface glycosaminoglycans.
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Herpes simplex virus type 2 UL34 protein requires UL31 protein for its relocation to the internal nuclear membrane in transfected cells
More LessHerpes simplex virus type 2 UL34 protein is expressed late in infection and is required for envelopment of nucleocapsids at the nuclear membrane and possibly at the endoplasmic reticulum (ER). It is a type II membrane protein with a C-terminal anchor that localizes mainly to the nuclear membrane in infected cells. However, in single transient expression, UL34 protein localizes predominantly to the ER. Relocation of UL34 protein from the ER to the internal nuclear membrane and the nucleus was observed in the presence of UL31 protein, a phosphoprotein known to interact physically with UL34. It is suggested here that interaction with UL31 protein is important for the nuclear targetting of UL34 protein and also that the trans-membrane region of UL34 protein is responsible for its localization at the internal nuclear membrane. The results also suggest possible sites for the interaction.
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In vitro selection of human cytomegalovirus variants unable to transfer virus and virus products from infected cells to polymorphonuclear leukocytes and to grow in endothelial cells
Four human cytomegalovirus (HCMV) isolates from different clinical sources were extensively propagated in human embryonic lung fibroblasts (HELF). Plaque isolates from each of the four virus strains were evaluated for their ability to be transferred to polymorphonuclear leukocytes (PMNL) and to grow in endothelial cells (EC). While all four of the clinical strains were found to be both PMNL- and EC-tropic, variants were identified from each of the four strains that lacked both biological properties, while three of the four parental strains lost their transfer capacity before passage 50 in HELF. It was demonstrated that one of the four field isolates (VR6110) and its transfer-deficient variant were genetically related, but showed different curves of virus yield in HELF. In addition, neither the immediate-early (IE) mRNA nor the IE protein p72 were found to be transferred to PMNL before 72 h post-infection (late in infection) or in the presence of viral DNA replication inhibitors. These findings link EC and PMNL tropism and suggest that PMNL tropism is a late HCMV function.
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Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97
The UL97-encoded protein kinase (pUL97) of human cytomegalovirus (HCMV) plays a critical role in the control of virus replication. Deletion of the UL97 gene results in a drastic reduction in the replication efficiency. Although the exact function of pUL97 remains unclear and its sensitivity to specific inhibitors is speculative, protein kinase inhibitors of the indolocarbazole class are effective inhibitors of cytomegalovirus. Based on the phosphorylation of ganciclovir (GCV), a novel quantification system for pUL97 kinase activity was established: the phosphorylated form of GCV exerts an easily quantifiable cytotoxic effect in transfected cells. Importantly, the addition of indolocarbazole compounds, Gö6976 and NGIC-I, which were highly effective at nanomolar concentrations while other protein kinase inhibitors were not, led to a significant reduction of pUL97 kinase activity. It was also demonstrated that a catalytically inactive mutant of pUL97, K355M, and a GCV-resistant mutant, M460I, were both negative for GCV phosphorylation, although protein phosphorylation remained detectable for the latter mutant. In vitro kinase assays were used to confirm the levels of pUL97-mediated phosphorylation recorded. To generate a tool for screening large numbers of putative inhibitors that preferentially interfere with GCV as well as protein phosphorylation, pUL97-expressing cell clones with stable pUL97 kinase activity were selected. This study demonstrates that certain indolocarbazole compounds are potent pUL97 inhibitors and, therefore, represent novel candidates for antiviral drugs that target viral protein kinase functions.
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Role of Epstein–Barr virus-encoded latent membrane protein 2A on virus-induced immortalization and virus activation
More LessTo quantitatively evaluate the role of Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in immortalization of peripheral B-lymphocytes, we used the Akata cell system to generate an EBV recombinant in which the first exon of the LMP2A gene was disrupted. The results indicated that deletion of the LMP2A gene did not affect the immortalization efficiency of EBV in B-lymphocytes. Deletion of the LMP2A gene made EBV-transformed lymphocytes more permissive for virus replication in response to surface immunoglobulin cross-linking. On the other hand Akata cells, in which LMP2A expression was much lower than in EBV-transformed lymphocytes, were equally permissive for virus replication whether they were infected with wild EBV or LMP2A-knockout EBV. The results raise a question as to the role of LMP2A in inhibition of disruption of virus latency in vivo, where LMP2A expression has been expected to be low as in Akata cells.
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Epstein–Barr virus-encoded protein kinase BGLF4 mediates hyperphosphorylation of cellular elongation factor 1δ (EF-1δ): EF-1δ is universally modified by conserved protein kinases of herpesviruses in mammalian cells
Translation elongation factor 1δ (EF-1δ) is hyperphosphorylated in various mammalian cells infected with alpha-, beta- and gammaherpesviruses and EF-1δ modification is mediated by viral protein kinases, including UL13 of herpes simplex virus type 1 and UL97 of human cytomegalovirus. In this study, the following is reported. (i) BGLF4 encoded by the prototype gammaherpesvirus Epstein–Barr virus was purified as a fusion protein that was labelled with [γ-32P]ATP and labelling was eliminated by phosphatase. (ii) The ratio of the hyperphosphorylated form of human EF-1δ was increased both in Sf9 cells after infection with baculoviruses expressing GST–BGLF4 fusion proteins and in COS-7 cells after transfection with a BGLF4 expression plasmid. These results indicate that purified BGLF4 possesses protein kinase activity and mediates EF-1δ hyperphosphorylation. These data also support the hypothesis that the protein kinases that are conserved by herpesviruses universally mediate EF-1δ modification in mammalian cells.
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Defining CAR as a cellular receptor for the avian adenovirus CELO using a genetic analysis of the two viral fibre proteins
More LessThe coxsackievirus and adenovirus receptor (CAR) is a high affinity receptor used by adenoviruses, including adenovirus type 5 (Ad5). The adenovirus fibre molecule bears the high affinity cell binding domain of Ad5, allowing virions to attach to CAR. The avian adenovirus CELO displays two fibre molecules on its capsid and it was logical to expect that the cell binding functions of CELO might also reside in one or both of these fibres. We had previously shown that the cell binding properties of CELO resemble Ad5, suggesting that the two viruses use similar receptors. Experiments with CAR-deficient CHO cells and CHO cells modified to express CAR demonstrated that CELO has CAR-dependent transduction behaviour like Ad5. Mutations were introduced into the CELO genome to disrupt either the long fibre 1 or the short fibre 2. A CELO genome with fibre 2 disrupted did not generate virus, demonstrating that fibre 2 is essential for some stage in virus growth, assembly or spread. However, a CELO genome with disrupted fibre 1 gene produced virus (CELOdF1) that was capable of entering chicken cells, but had lost both the ability to efficiently transduce human cells and the CAR-specific transduction displayed by wild-type CELO. The ability of CELOdF1 to transduce chicken cells suggests that CELOdF1 may still bind, probably via fibre 2, to a receptor expressed on avian but not mammalian cells. CELOdF1 replication was dramatically impaired in chicken embryos, demonstrating that fibre 1 is important for the in vivo biology of CELO.
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Cellular transcription factors that interact with p6 promoter elements of parvovirus B19
More LessAll transcripts of the human parvovirus B19 identified so far are regulated by a single promoter at map unit 6 of the viral genome, the so-called p6 promoter. This promoter is active in a wide variety of different cells. In order to identify cellular transcription factors involved in regulating promoter activity, we performed gel-retardation and supershift assays using the parts of the p6 promoter sequence shown previously to be protected in footprint experiments. Thereby, binding was demonstrated of the Oct-1 protein to an octamer motif within the p6 promoter and of the transcription factor Sp1 to three GC boxes. A specific preferential interaction of the factor Sp3 with one of these boxes was observed, indicating that the ratio Sp1:Sp3 may be involved in the regulation of promoter activity. Consensus sites for the regulatory protein YY1 are located close to the GC boxes and the octamer motif, to which this factor binds efficiently.
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- Plant
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Genes Ia, II, III, IV and V of Soybean chlorotic mottle virus are essential but the gene Ib product is non-essential for systemic infection
More LessSoybean chlorotic mottle virus (SbCMV) is the type species of the genus ‘Soybean chlorotic mottle-like viruses’, within the family Caulimoviridae. The double-stranded DNA genome of SbCMV (8178 bp) contains eight major open reading frames (ORFs). Viral genes essential and non-essential for the replication and movement of SbCMV were investigated by mutational analysis of an infectious 1·3-mer DNA clone. The results indicated that ORFs Ia, II, III, IV and V were essential for systemic infection. The product of ORF Ib was non-essential, although the putative tRNAMet primer-binding site in ORF Ib was proved to be essential. Immunoselection PCR revealed that an ORF Ia deletion mutant was encapsidated in primarily infected cells, indicating that ORF Ia is required for virus movement but not for replication. ORF IV was confirmed to encode a capsid protein by peptide sequencing of the capsid. Analysis of the viral transcripts showed that the SbCMV DNA genome gives rise to a pregenomic RNA and an ORF VI mRNA, as shown in the case of Cauliflower mosaic virus.
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Use of a vector based on Potato virus X in a whole plant assay to demonstrate nuclear targeting of Potato spindle tuber viroid
More LessPotato spindle tuber viroid (PSTVd) is a covalently closed circular RNA molecule of 359 nucleotides that replicates within the nucleus of host cells. To determine how this small, highly structured RNA enters the nucleus, we have developed a virus-based, whole plant in vivo assay that uses green fluorescent protein (GFP) as the reporter molecule. The coding region of GFP was interrupted by insertion of an intron derived from the intervening sequence 2 of the potato ST-LS1 gene. A cDNA copy of the complete PSTVd genome was, in turn, embedded within the intron, and this construct was delivered into Nicotiana benthamiana plants via a vector based on Potato virus X. The intron-containing GFP subgenomic RNA synthesized during virus infection cannot produce a functional GFP unless the RNA is imported into the nucleus, where the intron can be removed and the spliced RNA returned to the cytoplasm. The appearance of green fluorescence in leaf tissues inoculated with constructs containing a full-length PSTVd molecule embedded in the intron indicates that nuclear import and RNA splicing events did occur.
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A comparison of the solution structures of tobacco rattle and tobacco mosaic viruses from Raman optical activity
More LessVibrational Raman optical activity (ROA) spectra of tobacco rattle virus (TRV) and tobacco mosaic virus (TMV) were measured and compared with a view to obtaining new information about the coat protein subunit structure of TRV. A sharp strong positive band observed at ∼1344 cm−1 in the ROA spectra of the two viruses is evidence that both contain a significant amount of a hydrated form of α-helix, but more in TRV than in TMV. Although the ROA spectrum of TMV shows significant positive intensity in the range ∼1297–1312 cm−1 characteristic of α-helix in a hydrophobic environment, as expected from the helix interface residues in the four-helix bundles that constitute the basic motif of the TMV coat protein fold, that of TRV shows little positive ROA intensity here. Instead TRV shows a strong positive ROA band at ∼1315 cm−1, of much greater intensity than bands shown here by TMV, that is characteristic of polyproline II (PPII) helix. This suggests that the additional long central and C-terminal sequences of the TRV coat proteins contain a significant amount of PPII structure, plus perhaps some β-strand judging by a prominent sharp negative ROA band shown by TRV at ∼1236 cm−1, but little α-helix. The open flexible hydrated nature of PPII helical structure is consistent with the earlier suggestions that the additional sequences are exposed and, together with a larger amount of hydrated α-helix, could serve to fill the extra volume required by the larger diameter of the cylindrical TRV particles relative to those of TMV.
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Visualization by atomic force microscopy of tobacco mosaic virus movement protein–RNA complexes formed in vitro
More LessThe structure of complexes formed in vitro by tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short (890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on a mica surface. MP molecules were found to be distributed along the chain of RNA and the structure of MP–RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60–100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive ‘beads-on-a-string’ into a ‘thick string’ form that was partly resistant to RNase. The ‘thick string’-type RNase-resistant complexes were also produced by short synthetic RNA transcripts at different MP:RNA ratios. The ‘thick string’ complexes are suggested to represent clusters of MP molecules cooperatively bound to discrete regions of TMV RNA and separated by protein-free RNA segments.
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Nucleotide sequence of the coat protein gene of Lettuce big-vein virus
More LessA sequence of 1425 nt was established that included the complete coat protein (CP) gene of Lettuce big-vein virus (LBVV). The LBVV CP gene encodes a 397 amino acid protein with a predicted M r of 44486. Antisera raised against synthetic peptides corresponding to N-terminal or C-terminal parts of the LBVV CP reacted in Western blot analysis with a protein with an M r of about 48000. RNA extracted from purified particles of LBVV by using proteinase K, SDS and phenol migrated in gels as two single-stranded RNA species of approximately 7·3 kb (ss-1) and 6·6 kb (ss-2). After denaturation by heat and annealing at room temperature, the RNA migrated as four species, ss-1, ss-2 and two additional double-stranded RNAs (ds-1 and ds-2). The Northern blot hybridization analysis using riboprobes from a full-length clone of the LBVV CP gene indicated that ss-2 has a negative-sense nature and contains the LBVV CP gene. Moreover, ds-2 is a double-stranded form of ss-2. Database searches showed that the LBVV CP most resembled the nucleocapsid proteins of rhabdoviruses. These results indicate that it would be appropriate to classify LBVV as a negative-sense single-stranded RNA virus rather than as a double-stranded RNA virus.
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Mutational analysis of the proteinase function of Potato leafroll virus
More LesscDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513–515 of the replicase to VHD abolished PLRV replication. Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein. Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other. Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif.
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The ORF0 product of Potato leafroll virus is indispensable for virus accumulation
Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 of Potato leafroll virus (PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.
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Volume 105 (2024)
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